Harnessing snake venom cardiotoxins for antimicrobial peptide discovery

Abstract Snake venoms are a rich source of bioactive molecules with considerable potential for drug discovery. Cardiotoxins (CTXs) from cobras ( Naja spp.) are membrane-active venom proteins and promising templates for antimicrobial peptide development. Here, we combined two computational toxin-mining strategies to identify antimicrobial peptide candidates from CTXs. First, AMPA-guided sequence mining was used to detect encrypted antimicrobial regions within CTX sequences. Second, sequence alignment and consensus-sequence analysis were employed to generate peptides from a conserved CTX scaffold. Candidate peptides were prioritised using multiple machine learning- and deep learning-based antimicrobial peptide prediction tools, resulting in fourteen CTX-inspired peptides, including two modified derivatives of CTX-p5. Antimicrobial screening showed limited activity across the peptide panel, with CTX-p5 exhibiting the strongest activity against both Gram-negative and Gram-positive bacteria. Neither truncation nor chemical modification improved its antibacterial potency. Mechanistic studies, including atomic force microscopy, flow cytometry, fluorescence microscopy and membrane permeabilisation assays, indicated that CTX-p5 disrupts bacterial membrane integrity. These findings identify CTXs as a source of antimicrobial peptide templates while highlighting the limitations of current computational prioritisation approaches and the need for more accurate toxin-mining workflows to enhance peptide selection during toxin-inspired drug discovery.